DNA metabolism Flashcards
who did the experiment that proved how DNA is replicated
Meselson and Stahl
how did Meselson and Stahl show how DNA was replicated (ie by which method)
by tracing nitrogen isotopes through several generations of DNA replication
list the 3 proposed models of DNA replication
conservative, dispersive, semiconservative
describe conservative replication in the first send second rounds of replication
first: one strand is all heavy, other is all light
second: heavy strand produces one all heavy and one all light. Light strand only produces light
describe dispersive replication in the first send second rounds of replication
first: both strands produced are hybrids of heavy and light
second: all products have heavy dispersed in like a puzzle piece
describe semiconservative replication in the first send second rounds of replication
first: both products have one heavy and one light strand
second: each first gen produces one all light and one hybrid
what sized bands did meselson and stahl find throughout their experiment
original DNA = heavy
first gen = hybrid
second gen = light and hybrid
why did meselson and stahl’s experiment disprove the conservative model of replication
if it was conservative, there would be no hybrid bands observed; only heavy and light
why did meselson and stahl’s experiment disprove the dispersive model of replication
in the second gen, there would have been no light band if it was dispersive; only hybrid
where does replication begin
at an origin/replication fork where to strands separate
T or F: each strand of DNA is replicated one at a time
false; both are replicated simultaneously
which direction are DNA strands replicated
5’-3’
describe the number of replication origins in prokaryotes vs eukaryotes
prokaryotes: 1
eukaryotes: can have multiple
origins of replication are usually high in ___ base pairs
A-T
why are origins of replication high in AT pairs
AT pairs have 2 hydrogen bonds instead of 3 (like GC), so they’re easier to separate
by which method can regions with lots of AT pairs be found
denaturation mapping
describe denaturation mapping
increase the heat to make sequences rich in AT base pairs separate
which strand is synthesized in the same direction that the replication fork is moving
leading strand
briefly, how would one synthesize the lagging strand
synthesized in short 100-200bp okazaki fragments
how long are okazaki fragments
100-200bp
what is the DNA replication equation
(dNMP)n + dNTP → (dNMP)n+1 + PPi
what does the DNA replication equation mean
you have an existing strand, an NTP comes in and adds to it, giving us an elongated strand and a free pyrophosphate is released
describe the general structure of polymerase
has 2 Mg2+ residues and some Asp in the active site
describe the mechanism of polymerase as a primer comes in
the top Mg2+ of the enzyme deprotonates the 3’ OH of the primer to make it a more effective nucleophile. this facilitates attack of the 3’ OH on the alpha phosphate of the incoming NTP
The bottom Mg2+ helps displace the pyrophosphate. The Asp residues in the DNA pol active site bind Mg2+ and they’re highly conserved
during the polymerase mechanism, which part of the primer is deprotonated by the enzyme
3’ OH
which Mg2+ displaces pyrophosphate
the bottom one
during the polymerase mechanism, which type of bond is formed
phosphodiester
during the polymerase mechanism, which type of bond is cleaved
phosphoanhydride
T or F: growing a DNA strand via polymerase costs lots of free energy (G)
false; it doesn’t change the free energy that much
why is lengthened DNA more stable than a free dNTP
due to base stacking and base pairing
after pyrophosphate is displaced during strand elongation, what happens to it
it’s cleaved into two inorganic phosphates by inorganic pyrophosphatases
cleaving of PPi into two Pi releases how much energy
19 kJ/mol
what are the two parts of the polymerase active site
insertion and postinsertion sites
describe how the insertion/postinsertion sites in the polymerase active site function
once the phosphodiester bond is formed on the growing strand, the polymerase slides forward and the new base pair moves from the insertion site to the postinsertion site
once a nucleotide has been added to a growing strand, list the two things the polymerase can now do
it can move along the template or dissociate
define processivity
how many nucleotides a polymerase adds before dissociating
relate replication speed to processivity
replication is faster when processivity is higher
describe how the polymerase active site is resistant to errors
the active site only fits Watson-crick base pairs, so even if an incorrect base pair were to hydrogen bond with the template, it wouldn’t fit in the active site
describe polymerase proofreading mechanism
done by a separate active site. A mismatch impedes translocation to the next site, so the polymerase repositions the mismatch into an exonuclease active site. Mismatch is then removed and the polymerase converts to the original position to add the correct nucleotide
which pol is the main replication enzyme
pol III
which pol has the highest processivity and fastest polymerization rate
pol III
which type of activity do all pol have
3’-5’ exonuclease activity (for proofreading)
which type of pol has 5’-3’ exonuclease activity
pol I (= nick translation)
which subunits does the core pol III have
a, e, and theta
by which structure can two or three core polymerases be linked together
a clamp loading complex
which subunits encircle DNA and act like clamps to prevent the core from dissociating from DNA
beta (dimers)
role of clamp loader
links core polymerases together, binds ATP = conf change that allows DNA to slide into the b-clamp dimer
role of the core
this is where the actual replication/elongation occurs
role of the b-clamps
holds DNA in the core
T or F: b-clamp opens after ATP hydrolysis
false; ATP binds = clamp opens, ATP hydrolyzed = clamp closes
what is the replisome
DNA pol + all the other 20+ proteins involved in DNA replication
what is helicase
acts ahead of polymerase to separate the strands to be replicated
which molecule is helicase dependent on
ATP
what does SSB stand for
single stranded binding protein
what are SSBs
they stabilize DNA strands
what do topoisomerases do
they relieve structural strains generated by helicase
what do primases do
generate short RNA sections to serve as template primers
which enzyme removes RNA primers and replace them with DNA
DNA pol I
what does ligase do
seals the nicks remaining after pol I activity (pol I activity = replacing primers w/ DNA)
list the key sequences involved in initiation
DUE and 5 R sites
what does DUE stand for
DNA unwinding element
what is the DUE
a region rich in AT pairs (has 3 13-bp consensus sequences) near the origin of replication
what are the 5 R sites for
they’re consensus sequences bound by a key initiator protein DnaA
which protein binds to each R site (in initiation)
DnaA
what type of molecule is DnaA
an ATPase
how many DnaA molecules bind to the R sites
8
T or F: DnaA family of ATPases oligomerize
true
what affect does DnaA have
causes DNA to positively supercoil around the DnaA oligomers, inducing strain –> AT-rich DUE region separates
does DNA positively or negatively supercoil around the DnaA oligomers during initiation
positive
other than DnaA, what other ATPases are involved in initiation
DnaB and DnaC
describe how DnaB/C are involved in initiation
DnaC-ATP hexamer loads a DnaB hexamer onto each of the exposed strands. DnaC hydrolyzes its ATP and leaves, leaving the DnaB hexamer behind. DnaB will encircle each exposed strand
DnaB is also known as ___
helicase
describe how DnaB/helicase acts as an anchor during replication
other replisome proteins are linked directly or indirectly to it
when will DnaA hydrolyze its ATP
only after DNA pol III and the associated b-clamps have been loaded adjacent to the helicase
describe all the steps that occur during initiation
8 DnaA bind to R sites near the origin of replication. DNA positively supercoils around DnaA oligomers = strain = strands separate. DnaC-ATP loads DnaB onto each exposed strand. DnaC hydrolyzes its ATP and leaves, leaving DnaB behind to encircle each strand. Once pol III and the clamps have been loaded adjacent to DnaB, DnaA will hydrolyze its ATP and leave. Elongation can now occur
what is the rate of elongation
2000 nucleotides/sec
describe the actions of primase during elongation
it attaches to helicase and synthesizes a primer in the opposite direction to it’s unwinding
what does primase attach to during elongation
helicase (DnaB)
T or F: primase synthesizes a primer in the same direction of DNA unwinding
false; it synthesizes it in the opposite direction
describe how DNA pol III interacts with the primer during elongation
it binds to the primer and deoxyribonucleotides will be added
T or F: the leading and lagging strands are produced by the same pol III enzyme
true
since both strands are synthesized by the same pol III enzyme, what must happen to the structure of the unwound DNA?
lagging strand must be looped to bring both points of polymerization together near the 3 core domains
describe the actions of all 3 core domains during elongation
1 core domain is used continuously for the leading strand. The other 2 will leapfrog between each okazaki fragment
T or F: both leading and lagging strand are copied in the same 5’-3’ direction
true
which direction are the DNA strands copied in
5’-3’
during elongation, what causes a b-clamp to fall off
clamp falls off when the core subunits of DNA pol dissociate from the clamp
which DNA pol replaces the RNA primer with DNA
pol I
which type of activity does DNA pol I have that replaces RNA primer with DNA
5’-3’ exonuclease activity
T or F: pol I 5’-3’ exonuclease activity removes the nicks
false; it shifts the position open nick but does not remove it
after DNA pol I 5’-3’ exonuclease activity to get rid of primers, how do the nicks become sealed (by which enzyme)
ligase
describe the mechanism of ligase to seal nicks in DNA
AMP is added to ligase. Swap that AMP onto the exposed 5’ end of the nick. The exposed 3’ end of the nick attacks the alpha phosphate, displaces the AMP, and seals the nick
in termination, which two things must bind in order for it to occur
a Ter sequence binding to a TUS protein
what does TUS stand for
terminus utilization substance
how long are Ter sequences
20 bp
what happens once the TUS protein is bound to a Ter sequence
it will arrest the first replication fork that it runs into
in termination, what happens once the first replication fork is arrested
the second fork, coming from the other direction eventually halts when it runs into the first arrested fork
what happens when replication forks run into each other and are arrested
they fall off at the Ter sequence
what is left over after replication forks are arrested and fall off at the Ter sequence
there are still a few hundred bp that need to be replicated still
during termination, how are the leftover bp replicated?
by an unknown mechanism
T or F: in termination, even when fully replicated the strands are still topologically linked
true!
T or F: fully replicated DNA strands have a linking number
true!
what are fully replicated, linked DNA strands called
catenanes
what are catenanes
fully replicated DNA that are still topologically linked and have a linking number
which enzyme cuts catenanes (linked + fully replicated DNA) and separates the strands
topoisomerase II family member
what do topoisomerase II family members do during termination
they separate the linked DNA strands (catenanes) for fully separated chromsomes
